Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

Maternal protein restriction during pregnancy affects gene expression and immunolocalization of intestinal nutrient transporters in rats

Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.

Gene expression and protein localization of TLR-1, -2, -4 and -6 in amniochorion membranes of pregnancies complicated by histologic chorioamnionitis

Objectives: To determine whether histologic chorioamnionitis is associated with changes in gene expression of TLR-1, -2, -4 and -6, and to describe the localization of these receptors in fetal membranes. Study design: A total of 135 amniochorion membranes with or without histologic chorioamnionitis from preterm or term deliveries were included. Fragments of membranes were submitted to total RNA extraction. RNA was reverse transcribed and the quantification of TLRs expression measured by real time PCR. Results: All amniochorion membranes expressed TLR-1 and TLR-4, whereas 99.1% of membranes expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expressions were significantly higher in membranes with histologic chorioamnionitis as compared to membranes without chorioamnionitis in preterm pregnancies (p = 0.003 and p < 0.001, respectively). Among the membranes of term pregnancies there were no differences in the expressions of such receptors regardless of inflammatory status. Regarding TLR-4 and TLR-6 expression, there was no difference among membranes with or without histologic chorioamnionitis, regardless gestational age at delivery. TLR-1, TLR-2, TLR-4 and TLR-6 expressions were observed in amniotic epithelial, chorionic and decidual cells. Conclusion: Amniochorion membranes express TLR-1, TLR-2, TLR-4 and TLR-6 and increased expression of TLR-1 and TLR-2 is related to the presence of histologic chorioamnionitis in preterm pregnancies. This study provides further evidence that amniochorion membranes act as a mechanical barrier to microorganisms and as components of the innate immune system. © 2013 Elsevier B.V. All rights reserved.

Pesquisa de mutação do gene predito da lectina ligante de manose em cães: comparação entre dois alvos para diagnóstico molecular de leishmaniose visceral canina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolamento, caracterização molecular e expressão de um novo gene vip3Aa50 de Bacillus thuringiensis virulento para Anticarsia gemmatalis e Spodoptera frugiperda

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Novo gene cry8 de Bacillus thuringiensis: caracterização e avaliação da patogenicidade ao bicudo da cana-de-açúcar, Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização molecular do gene que codifica a TCTP (Translationally Controlled Tumor Protein) em tomate (Solanum lycopersicum cv. Santa Clara)

The gene encoding TCTP (Translationally Controlled Tumour Protein) is present in all eukaryotes and its product is involved in various cellular processes. Although well characterized in mammals, there are only few works available in the literature related to the analysis of this protein in plants. In this present work, the expression of the gene encoding TCTP was analyzed in different organs/tissues of tomato plants (Solanum lycopersicum cv. Santa Clara). A quantification performed by RT-qPCR revealed the presence of TCTP transcript in all tissues/organs analyzed, with the highest expression level found in leaves. With the exception of fruits in intermediate stage of maturation, for which a small increase on the expression was detected, there was minimal variation in the relative expression of TCTP in other organ/tissues. In parallel, the effects of the constitutive expression of TCTP were investigated using transgenic tobacco lines able to overexpress this protein at different levels (T1, T2 and T3). Seedlings of these lines, and of a non-transgenic control line, were grown in MS culture medium for 21 days. At the end of this period, the length of roots and leaves was taken and the seedlings were photographed. According to Tukey's test, the analysis of the mean root length revealed a significant difference between T1 and T3 lines when compared to the control, although the same was not observed for the T2 line. For leaves, according to Kruskal-Wallis test, there was a statistical difference between the averages of leaf growth obtained for the different lines evaluated. According to these results, we can conclude that TCTP shows an ubiquitous expression in tomato plants, with the highest expression detected in leaves, and also that its overexpression promoted a higher root and leaf development in two of three transgenic tobacco lines tested

Gene and protein expression of oestrogen-beta and progesterone receptors in facial melasma and adjacent healthy skin in women

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Experimental glaucoma model (ischemia and reperfusion): histology, morphometry, protein and gene espression of apoptosis pathway

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kaurenoic Acid from Sphagneticola trilobata Inhibits Inflammatory Pain: Effect on Cytokine Production and Activation of the NO-Cyclic GMP-Protein Kinase G-ATP-Sensitive Potassium Channel Signaling Pathway

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-alpha and IL-1 beta. Furthermore, the analgesic effect of 1 was inhibited by L-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein lcinase G-ATP-sensitive potassium channel signaling pathway.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

Haplotypes of susceptibility to chronic periodontitis in the Interleukin 8 gene do not influence protein level in the gingival crevicular fluid

Objective: Previously, we identified that the ATC/TTC haplotype formed by polymorphisms in the Interleukin-(IL)8 gene conferred susceptibility to chronic periodontitis (CP). The aim of the study was to investigate whether the IL8 haplotype ATC/TTC was associated with the volume of gingival crevicular fluid (GCF), the concentration of interleukin IL-8 in the GCF, as well as periodontal conditions in patients with CP in comparison to controls without CP. Methods: Seventy-nine individuals (CP: n = 41, controls: n = 38) were grouped according to the presence (susceptible for CP) or absence (not susceptible for CP) of the IL8 ATC/TTC haplotype. After periodontal clinical evaluation, they were subdivided by the presence or absence of CP. GCF was collected from each patient and the IL-8 levels were determined by ELISA. The GCF volume of each subject was measured by means of a calibrated electronic device. Comparisons of means between carriers and non-carriers of the ATC/TTC haplotype were evaluated using the Mann-Whitney test. Linear regression and stepwise linear regression analysis were used to analyse the association of the GCF volume with potential covariates and their contribution for the phenotype. Results: We did not find significant differences of both periodontal conditions and IL-8 concentration in the GCF of patients with the presence or absence of the IL8 ATC/TTC haplotype. However, the GCF volume was significantly higher amongst the patients affected by CP that are absent for the IL8 ATC/TTC haplotype. In addition, linear regression analysis showed a statistically significant association between GCF volume and CP, IL8 haplotype ATC/TTC and IL-8 concentration. Conclusions: The IL8 haplotype of susceptibility to CP was neither associated with IL-8 cytokine levels nor with clinical periodontal parameters. Also, CP, IL8 haplotype and IL-8 concentration showed a positive association with the GCF volume levels in the studied patients. (c) 2012 Published by Elsevier Ltd.

Prognostication of Soft Tissue Sarcomas Based on Chromosome 17q Gene and Protein Status: Evaluation of TOP2A, HER-2/neu, and Survivin

Topoisomerase 2 alpha (), HER-2/ and are genes that lie on chromosome 17 and correlate with the prognosis and prediction of target-driven therapy against tumors. In a previous study, we showed that TOP2A transcripts levels were significantly higher in soft tissue sarcomas (STS) than in benign tumors and desmoid-type fibromatoses (FM). Because these genes have been insufficiently examined in STS, we aimed to identify alterations in TOP2A and HER-2 expression by fluorescent in situ hybridization and immunohistochemistry, as well as that of survivin, and correlate them with clinicopathologic findings to assess their prognostic value. Eighteen FM and 244 STS were included. Fluorescent in situ hybridization and immunohistochemistry were performed on a tissue microarray. TOP2A and survivin were more highly expressed in sarcomas than in FM. TOP2A was an independent predictor of an unfavorable prognosis; it was combined with formerly established prognostic factors (primarily histologic grade and tumor size at diagnosis) to create a prognostic index that evaluated overall survival. Gene amplification/polysomy (13%) did not correlate with protein overexpression. Survivin and HER-2 expression were not associated with patient outcomes. These findings might become valuable in the management of patients with STS and possibly in the prospective evaluation of responses to new target-driven therapies.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors

Aims: To evaluate the associations of excision repair cross complementing-group 1 (ERCC1) (DNA repair protein) (G19007A) polymorphism, methylation and immunohistochemical expression with epidemiological and clinicopathological factors and with overall survival in head and neck squamous cell carcinoma (HNSCC) patients. Methods and results: The study group comprised 84 patients with HNSCC who underwent surgery and adjuvant radiotherapy without chemotherapy. Bivariate and multivariate analyses were used. The allele A genotype variant was observed in 79.8% of the samples, GG in 20.2%, GA in 28.6% and AA in 51.2%. Individuals aged more than 45 years had a higher prevalence of the allelic A variant and a high (83.3%) immunohistochemical expression of ERCC1 protein [odds ratio (OR) = 4.86, 95% confidence interval (CI): 1.2-19.7, P = 0.027], which was also high in patients with advanced stage (OR= 5.04, 95% CI: 1.07-23.7, P = 0.041). Methylated status was found in 51.2% of the samples, and was higher in patients who did not present distant metastasis (OR = 6.67, 95% CI: 1.40-33.33, P = 0.019) and in patients with advanced stage (OR = 5.04, 95% CI: 1.07-23.7, P = 0.041). At 2 and 5 years, overall survival was 55% and 36%, respectively (median = 30 months). Conclusion: Our findings may reflect a high rate of DNA repair due to frequent tissue injury during the lifetime of these individuals, and also more advanced disease presentation in this population with worse prognosis.